This is a revised application for a Fogarty International Research Collaboration Award (FIRCA). The PI of the application is Dr. Teresita Bellido from the Center of Osteoporosis and Metabolic Bone Diseases of the University of Arkansas for Medical Sciences, USA. The foreign collaborator is Dr. Ricardo Boland from the Department of Biology, Biochemistry and Pharmacy of the Universidad Nacional del Sur, Argentina. The work proposed is an extension of an NIH-funded "parent grant" lead by the PI (see under Performance Site) and will be primarily performed in Argentina taking advantage of the expertise of the foreign collaborator. Studies by Dr. Bellido's laboratory demonstrated that bisphosphonates (BPs) prevent osteocyte and osteoblast apoptosis in vitro and in vivo, by a novel mechanism that involves the opening of hemichannels containing connexin43 (Cx43) (but not other connexins), with the subsequent activation of Src kinases, followed by activation of the extracellular signal regulated kinases (ERKs). Studies from Dr. Boland's laboratory demonstrated the presence of specific binding sites for the BP olpadronate in ROS17/2.8 rat osteosarcoma derived cells. ERKs can be activated by a variety of stimuli through the engagement of appropriate receptors, however receptors for BPs have not been described. Based on the lines of evidence from both laboratories, the PI proposes the hypothesis that opening of Cx43 hemichannels and subsequent activation of intracellular signaling is initiated by binding of bisphosphonates to extracellular domains of Cx43. To test this hypothesis, the PI and her collaborator propose the following specific aims to be completed in a three-year funding period. Specific Aim 1: Characterize the binding properties of the BP alendronate to Cx43-expressing cells, by determining the affinity constant (Kd), number of binding sites (Nmax), and binding specificity by competition analysis. Specific Aim 2: Map the domains of Cx43 involved in the BP binding by blocking specific regions of Cx43 using specific antibodies and blocking peptides. Specific Aim 3: Examine whether, similar to the anti-apoptotic actions of BPs mediated by ERKs, BPs activate other intracellular signaling cascades through Cx43 hemichannels that are responsible for the proliferative and differentiating effects of these agents on cells of the osteoblastic lineage. Funding of this FIRCA will allow the PI to answer an important scientific question in a timely manner, will foster research of high scientific merit beneficial for both the PI and her foreign collaborator, and will enhance the research capabilities at the foreign site.